Recent evidence suggests that cancers have a cellular hierarchy in which a minority population of cancer cells, called cancer stem cells, drives the growth and spread of a tumor. The ability to prospectively identify tumorigenic cancer cells will facilitate the identification of pathways that regulate their growth, metastasis and survival in patients. Targeting the pathways involved in essential processes such as self renewal may lead to more effective therapies. Preliminary evidence demonstrates that there is a host of genes differentially expressed by the cancer stem cells and their non-tumorigenic progeny. Many of these genes, both mRNAs encoding critical proteins and non-coding micro RNAs thought to modulate gene expression/translation, are thought to play a role in essential cancer functions including proliferation, survival, self renewal and resistance to standard therapeutics. Our studies are designed first to identify new and better therapeutic targets and then to identify drugs against these targets. They will rely on both in vivo (xenograft assays) and in vitro (microfluidic technology for single cell gene expression, chromatin immunoprecipitation and cell culture) approaches, adapted to the challenging particularities of cancer stem cells (rarity and primary origin). The following aims will allow us to accomplish this goal. Specific Aim 1. To identify drugs that target critical cancer stem cell targets. Specific Aim 2. To determine the mechanisms which regulate reactive oxygen species (ROS) in cancer stem cells. Specific Aim 3. To identify other cancer stem cell therapeutic targets. All 3 aims will be a collaborative effort with both Projects 1 and 3 and will heavily utilize the animal and flow cytometry cores. Aims 2 and 3 will also require the bioinformatics core for success